Recombinant Human B3GalT5 His-tag Protein, CF SummaryLearn more about Fluorescent Glycan Labeling and Detection
Asn29-Val310, with a C-terminal 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, pH 7.5
- Glycosyltransferase Activity Kit (Catalog # EA001)
- Recombinant Human B3GalT5 (rhB3GalT5) (Catalog # 10555-GT)
- Donor Substrate: UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
- Acceptor Substrate: GlcNAc (N-Acetyl-alpha -D-glucosamine) (Millipore, Catalog # 1079), 1 M stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
- Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmoles per well.
- Prepare reaction mixture containing 0.4 mM UDP-Galactose, 200 mM GlcNAc and 4 µg/mL Coupling Phosphatase 1 (supplied in kit) in Assay Buffer.
- Dilute rhB3GalT5 to 8 µg/mL in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 25 µL of 8 µg/mL rhB3GalT5 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
- Add 25 µL of reaction mixture to all wells, excluding the standard curve.
- Seal plate and incubate at 37 °C for 20 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|Specific Activity (pmol/min/µg) =||Phosphate released* (nmol) x (1000 pmol/nmol)|
|Incubation time (min) x amount of enzyme (µg)|
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.Per Reaction:
- rhB3GalT5: 0.2 µg
- Coupling Phosphatase 1: 0.1 µg
- UDP-Galactose: 0.2 mM
- GlcNAc: 100 mM
2 μg/lane of Recombinant Human B3GalT5 His-tag Protein (Catalog # 10555-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 40-45 kDa.
B3GalT5 is a glycosyltransferase in the beta-1,3-galactosyltransferase (B3GalT) family that contains 5 members. It is synthesized as type II membrane-bound glycoproteins in the Golgi apparatus (1). The enzyme catalyzes the synthesis of type 1 lactosamine structure (Gal beta 1-3GlcNAc) in type 1 Lewis antigens (Lea and Leb). Lea and Leb are elevated in gastrointestinal and pancreatic cancers (2). Sialylated Lea (Neu5Ac alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta ), also known as CA19-9, is a tumor marker in the gastrointestinal tract (Stomach, colon, and pancrease) (3). Down-regulation of B3GalT5 will reduce the level of CA19-9 (4, 5). In contrast, beta-1,4-galactosyltransferases synthesize type 2 lactosamine structure (Gal beta 1-4GlcNAc) that is found in type 2 Lewis antigens (Lex and Ley). B3GalT5 is also known as SSEA-3 synthase and synthesizes the structure Gal beta 1-3GalNAc in stage-specific embryonic antigen-3 (SSEA-3) antigen (6). SSEA-3 plays a key role in identifying many types of mammalian cells with pluripotent and stem cell-like characteristics (7). It also catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the Core-3 O-linked glycan GlcNAc beta 1-3GalNAc structure (8). The activity of this enzyme has been measured with a phosphatase-coupled method (9).
- Isshiki S. et al. (1999) J. Biol. Chem. 274:1249.
- Holgersson J and Löfling J. (2006) Glycobiology 16:584.
- Magnani, JL (2004). Arch. Biochem. Biophys. 426:122.
- Mare L. and Trinchera M. (2004) Eur. J. Biochem. 271:186.
- Zulueta, A.et al. (2014) FASEB J. 28:946.
- Zhou D. et al. (2000) J. Biol. Chem. 275:22631.
- Kuroda, Y. et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107: 8639.
- Zhou D. et al. (1999) Eur. J. Biochem. 263:571..
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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